U.S. patent application number 17/415515 was filed with the patent office on 2022-02-10 for determination and quantification of proteose peptone content and/or beta-casein content and nutritional composition with reduced beta-casein derived proteose peptone content.
The applicant listed for this patent is SOCIETE DES PRODUITS NESTLE S.A.. Invention is credited to Michael Affolter, Peter Erdmann, Christophe Fuerer, Jonathan O'Regan.
Application Number | 20220042961 17/415515 |
Document ID | / |
Family ID | 1000005973833 |
Filed Date | 2022-02-10 |
United States Patent
Application |
20220042961 |
Kind Code |
A1 |
Affolter; Michael ; et
al. |
February 10, 2022 |
DETERMINATION AND QUANTIFICATION OF PROTEOSE PEPTONE CONTENT AND/OR
BETA-CASEIN CONTENT AND NUTRITIONAL COMPOSITION WITH REDUCED
BETA-CASEIN DERIVED PROTEOSE PEPTONE CONTENT
Abstract
The present invention relates to a method for determining and
quantifying .beta.-casein derived proteose peptones and/or
.beta.-casein, said method comprising the steps of (i) providing a
dairy-based product to be analysed; (ii) subjecting said product
using liquid chromatography-mass spectrometry analysis; (iii)
determining and/or quantifying said .beta.-casein derived proteose
peptones and/or .beta.-casein in said product by detecting
compounds of defined m/z values or deconvoluting the mass
spectrometry spectra to calculate monoisotopic masses. The present
invention also relates to nutritional compositions having a reduced
content of .beta.-casein derived proteose peptones and the uses
hereof for e.g. treating, preventing and/or ameliorating abdominal
pain in an infant.
Inventors: |
Affolter; Michael; (Savigny,
CH) ; Erdmann; Peter; (Bern, CH) ; Fuerer;
Christophe; (Etoy, CH) ; O'Regan; Jonathan;
(Killarney, IE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SOCIETE DES PRODUITS NESTLE S.A. |
Vevey |
|
CH |
|
|
Family ID: |
1000005973833 |
Appl. No.: |
17/415515 |
Filed: |
November 28, 2019 |
PCT Filed: |
November 28, 2019 |
PCT NO: |
PCT/EP2019/082863 |
371 Date: |
June 17, 2021 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/6848 20130101;
G01N 2333/4731 20130101; G01N 33/04 20130101; A23L 33/19
20160801 |
International
Class: |
G01N 33/04 20060101
G01N033/04; A23L 33/19 20060101 A23L033/19; G01N 33/68 20060101
G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 20, 2018 |
EP |
18214628.2 |
Claims
1. A method for determining and/or quantifying .beta.-casein
derived proteose peptones and/or .beta.-casein, the method
comprising the steps of (i) providing a dairy-based product to be
analysed; (ii) subjecting the product using liquid
chromatography-mass spectrometry analysis; and (iii) determining
and/or quantifying the .beta.-casein derived proteose peptones
and/or .beta.-casein in the product by detecting compounds of
defined m/z values or deconvoluting one or more mass spectrometry
spectra to calculate monoisotopic masses.
2. The method according to claim 1, wherein the .beta.-casein
derived proteose peptones are PP8 fast, PP8 slow and PP-5.
3. The method according to claim 1, wherein the product is analysed
by intact protein analysis.
4. The method according to claim 1, wherein the product is analysed
by peptide analysis comprising a step of enzymatic digest of the
product prior to analysing it.
5. (canceled)
6. A method for producing a whey protein fraction having a reduced
.beta.-casein derived proteose peptone content, the method
comprising the steps of (i) providing a whey protein fraction; and
(ii) reducing the .beta.-casein derived proteose peptone content in
the whey protein fraction to a concentration of at the most 10% by
weight based on total protein in the whey protein fraction forming
a reduced whey protein fraction.
7. The method according to claim 6, wherein the proteose peptone
content is reduced by gel filtration.
8. A method for producing a nutritional composition having a
reduced .beta.-casein derived proteose peptone content, the method
comprising the steps of a. providing a selected whey protein
fraction or a reduced whey protein fraction having a reduced
.beta.-casein derived proteose peptone content, using a method
comprising the steps of (i) providing a whey protein fraction; (ii)
determining and quantifying the .beta.-casein derived proteose
peptones in the whey protein fraction; and (iii) selecting the whey
protein fraction having at the most 10% by weight of .beta.-casein
derived proteose peptones based on the total protein in the whey
protein fraction forming a selected whey protein fraction; b.
preparing the nutritional composition with a reduced proteose
peptone content from the selected whey protein fractions, the
reduced whey protein fractions or a mixture hereof.
9-15. (canceled)
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to a method for determining
and quantifying .beta.-casein derived proteose peptones such as
PP-5, PP8s and PP8f, and/or .beta.-casein. The present invention
also relates to a nutritional composition and an infant formula
comprising a reduced content of .beta.-casein derived proteose
peptones such as PP-5, PP8s and PP8f.
BACKGROUND OF THE INVENTION
[0002] Milk from dairy cows has been regarded as nature's perfect
food, providing an important source of nutrients including high
quality proteins, carbohydrates and selected micronutrients. Milk
proteins are primarily caseins with the remaining proteins being
grouped into the group of whey proteins, the major of which are
beta-lactoglobulin and alpha-lactalbumin. Among the caseins,
.beta.-casein is the second most abundant protein in cow's milk and
has an excellent nutritional balance of amino acids. However,
different mutations in the bovine .beta.-casein gene have led to
several genetic variants where the so-called A1 and A2 variants are
the most common.
[0003] The A1 and A2 variants of .beta.-casein differ at amino acid
position 67 of the secreted .beta.-casein protein, with histidine
(His) in the A1 variant of .beta.-casein and proline (Pro) in the
A2 variant of .beta.-casein, as a result of a single nucleotide
difference. This is illustrated in FIG. 1.
[0004] The A2 variant of .beta.-casein is more comparable to human
.beta.-casein in terms of digestive breakdown. It has been
suggested that the A1 variant of .beta.-casein may be associated
with cow's milk intolerance, and a statistically significant
positive association between abdominal pain and stool consistency
has been observed when participants consumed an A1 diet, but not an
A2 diet (Ho et al, 2014).
[0005] The digestive difference observed between the A1 and A2
variants may be due to the particular polymorphism as described
above. The polymorphism leads to a key conformational change in the
secondary structure of the .beta.-casein protein.
[0006] Gastrointestinal proteolytic digestion of the A1 (but not
the A2) variant of .beta.-casein leads to generation of
.beta.-casomorphin 7 (BCM-7) that is an exogenous opioid peptide
(exorphin) that may activate opioid receptors throughout the body
(EFSA Scientific Report, 2009). This is illustrated in FIG. 2.
[0007] Originally, all cows produced milk containing only the A2
variant of .beta.-casein, but genetic variation has resulted in
mixed herds. Thus, cow herds that are typically used to produce
milk normally produce a mixture of the A1 and A2 variants of
.beta.-casein but with different amount of A1 as compared to
A2.
[0008] Proteolysis of .beta.-casein by plasmin has been shown to
lead to the generation of .gamma.-caseins and proteose peptones
(PP8 fast, PP8 slow and PP-5). The proteose peptones are not
precipitated with caseins during acidification or enzymatic
treatment with rennet and remain in the whey fraction (reviewed in
Karamoko et al. 2013). PP8s slow and PP-5 correspond to sequences
29-105/7 and 1-105/7, respectively, and both include position 67.
Thus, whey products derived from milk containing A1 .beta.-casein
will likely contain proteose peptones of the A1 type. The proteose
peptones are derived from the same gene and occur through
endogenous events, why they are also considered as proteoforms of
.beta.-casein.
[0009] Intact protein analyses by reverse-phase-high performance
liquid chromatography (RP-HPLC) or capillary electrophoresis (CE)
coupled to UV detection can effectively separate most .beta.-casein
variants in raw milk samples (see for instance de Jong et al.,
1993, Visser et al., 1995, Bonfatti et al., 2008, Poulsen et al.,
2016). In manufactured products however, processing conditions
induce reactions between proteins and reducing sugars (Maillard
reaction) that yield multiple proteoforms containing one or more
sugar adducts (Fenaille et al., 2006). As a consequence, protein
peaks split and broaden, creating overlaps that prevent the
analysis of single proteoforms using UV detection (Vallejo-Cordoba,
1997, Feng et al., 2017).
[0010] The human Breast Milk (HBM) is known to contain the A2 form
of human beta-casein. By definition, it represents a gold standard
in terms of infant nutrition. There is therefore a need to provide
(synthetic) nutritional compositions that mimic in the best
possible manner the composition of HBM.
[0011] Due to the digestive differences observed in humans toward
the A1 and A2 variants of cow .beta.-casein, it would be
advantageous to be able to detect and quantify in a simple and
efficient way the amount of .beta.-casein and its proteoforms.
Currently, there is no simple and efficient method to characterize
and quantify individual milk proteins and their proteoforms such as
proteose peptones in finished products.
[0012] Furthermore, there is a need to provide a nutritional
composition that comprises a relatively low amount or proportion of
A1 .beta.-casein or of .beta.-casein derived proteose peptones or
may be deprived of any form hereof.
[0013] Also, there is a need to provide a nutritional composition
that comprises whey fractions, which are deprived or reduced in
.beta.-casein derived proteose peptones.
SUMMARY OF THE INVENTION
[0014] An object of the present invention relates to an improved
method for efficient and simple detection of .beta.-casein and its
proteoforms in particular its .beta.-casein derived proteose
peptones as well as a method for reducing the content of
.beta.-casein derived proteose peptones.
[0015] It is a further object of the present invention to provide a
nutritional composition such as an infant formula, which may
prevent abdominal pain and/or improve stool inconsistency of an
infant.
[0016] Thus, one aspect of the invention relates to a method for
determining and quantifying .beta.-casein derived proteose peptones
and/or .beta.-casein, said method comprising the steps of [0017]
(i) providing a dairy-based product to be analysed; [0018] (ii)
subjecting said product using liquid chromatography-mass
spectrometry analysis; [0019] (iii) determining and/or quantifying
said .beta.-casein derived proteose peptones and/or .beta.-casein
in said product by detecting compounds of defined m/z values or
deconvoluting one or more mass spectrometry spectra to calculate
monoisotopic masses.
[0020] A second aspect of the invention relates to a method for
producing a whey protein fraction having a reduced .beta.-casein
derived proteose peptone content, said method comprising the steps
of [0021] (i) providing a whey protein fraction; [0022] (ii)
determining and quantifying said .beta.-casein derived proteose
peptones in said whey protein fraction as described herein; and
[0023] (iii) selecting said whey protein fraction having at the
most 10% by weight of .beta.-casein derived proteose peptones based
on the total protein in said whey protein fraction forming a
selected whey protein fraction.
[0024] A third aspect of the invention relates to a method for
producing a whey protein fraction having a reduced .beta.-casein
derived proteose peptone content, said method comprising the steps
of [0025] (i) providing a whey protein fraction; [0026] (ii)
reducing the .beta.-casein derived proteose peptone content in said
whey protein fraction to a concentration of at the most 10% by
weight based on total protein in the whey protein fraction forming
a reduced whey protein fraction.
[0027] A fourth aspect of the invention relates to a method for
producing a nutritional composition having a reduced .beta.-casein
derived proteose peptone content, said method comprising the steps
of [0028] (i) providing a selected whey protein fraction or a
reduced whey protein fraction as described herein; [0029] (ii)
preparing said nutritional composition with a reduced .beta.-casein
derived proteose peptone content from said selected whey protein
fractions, said reduced whey protein fractions or a mixture
hereof.
[0030] A fifth aspect of the invention relates to a whey protein
fraction having a reduced .beta.-casein derived proteose peptone
content, obtainable by the method as described herein.
[0031] A sixth aspect of the invention relates to a nutritional
composition comprising said whey protein fraction having a reduced
.beta.-casein derived proteose peptone content as described herein
or obtainable by the method as described herein.
[0032] A seventh aspect of the invention relates to a nutritional
composition as described herein for use as a medicament.
[0033] An eight aspect of the invention relates to an infant
formula as described herein for use in treating, preventing and/or
ameliorating abdominal pain in an infant and/or improving gut
comfort in said infant.
[0034] A ninth aspect of the invention relates to an infant formula
as described herein for use in treating, preventing and/or
ameliorating lactose intolerance in an infant.
[0035] A tenth aspect of the invention relates to the use of the
infant formula as described herein for improving the stool
consistency.
BRIEF DESCRIPTION OF THE FIGURES
[0036] FIG. 1 shows a partial sequence alignment between the
sequence of A2 beta-casein and A1 beta-casein relating to the amino
acid sequences 59-71. The amino acid at position 67 is highlighted
demonstrating the one amino acid difference between A1 beta-casein
and A2 beta-casein being His and Pro, respectively;
[0037] FIG. 2 shows a partial sequence alignment between the
sequence of A2 beta-casein and A1 beta-casein relating to the amino
acid sequences 59-71. The one amino acid difference between the two
beta-caseins influences the cleavage ability of the two strands as
A2 beta-casein is not cleaved due to the presence of Pro at
position 67, while A1 beta-casein is cleaved on the N-terminal side
of His forming a possibility for a seven amino acid long sequence
to be formed i.e. Beta-casomorphin-7 (BCM-7);
[0038] FIG. 3 shows a description of the LC-HRMS method steps: (a)
sample preparation, (b) deconvoluted spectra with monoisotopic
masses for intact .beta.-casein and (c) bubble chart representation
of the intact .beta.-casein region (bubble sizes represent signal
intensities).
[0039] FIG. 4 shows the global fingerprinting and the intact
.beta.-casein region. Major milk proteins were separated in a (a)
global fingerprint of a raw milk sample. Different proteoforms were
detected for (b) intact .beta.-casein. A1, A2 and B are genetic
variants, superscript notations correspond to the number of lactose
(L) adducts;
[0040] FIG. 5 shows examples of global fingerprinting in raw milk,
human milk, infant formula (IF), skim milk powders (SMP) and whey
samples;
[0041] FIG. 6 shows bubble charts (intact .beta.-casein region) of
seven A2 infant formula samples either untreated or spiked at 5%
with .beta.-casein A1 (5 g of intact .beta.-casein A1/100 g of
total intact .beta.-casein);
[0042] FIG. 7 shows how glycation affects MS readings. (a) Bubble
charts (intact .beta.-casein region) showed increased lactosylation
during solid-state glycation experiment. (b) The percentage of
unglycated signal (black bar) decreased over time (c) shows the
total .beta.-casein signal decreases with increased glycation and
(d) had a linear relationship with the percentage of unglycated
signal. Plotting the signal reduction as a function of the
percentage of unglycated signal (e) allowed calculating a glycation
correction factor;
[0043] FIG. 8 shows a characterization of the .beta.-casein
standard and establishment of the .beta.-casein calibration curve.
Analysis of the .beta.-casein standard revealed that about 15% of
total signal could not be attributed to intact .beta.-casein (a). A
linear calibration curve was established using normalized (to SMP,
which was corrected for glycation) intact .beta.-casein MS signal
and adjusted .beta.-casein amounts (b). Cross-mixing experiments
between A1|A1 and A2|A2 skim milk samples indicated that the
relationship remained linear even in a dairy matrix (c). The
protein content was kept constant during the cross-mixing
experiment;
[0044] FIG. 9 shows the quantification of intact .beta.-casein in
infant formulas. .beta.-casein was quantified in seven infant
formula samples (a), where each sample was analyzed in triplicate
five times. Theoretical values are based on recipe and generic milk
protein composition. A further set of infant formulas (both
A2-based or based on skim milk powder containing multiple genetic
variants) was analyzed in triplicate in a single day (b). Hatched
bars represent best guesses because the % whey of those samples was
not indicated on the boxes;
[0045] FIG. 10 shows the result of LC-HRMS analysis of intact
proteins in different whey protein concentrates (WPC). The intact
protein region is marked by the circle with the solid perimeter,
the .beta.-casein proteose peptones are included in the region
marked by the circle with the dashed perimeter;
[0046] FIG. 11 shows a close-up of the area shown in FIG. 10 by the
circle with the dashed perimeter demonstrating the content of
proteose peptones. Black represents .beta.-casein A2 derived
proteose peptones, gray represents .beta.-casein A1 derived
proteose peptones and white represent unassigned compounds;
[0047] FIG. 12 shows the result of LC-HRMS analysis of intact
proteins in different finished products focusing on the area
containing proteose peptones.
[0048] FIG. 13 shows the correlation between measured PP5 signal
for both PP5 A1 and PP5 A2 in relation to the percentage of A2 SMP
in the sample. The protein content was kept constant during the
cross-mixing experiment;
[0049] FIG. 14 shows a schematic view of intact .beta.-casein and
products generated upon .beta.-casein cleavage. The position of the
tryptic peptides are shown by the horizontal arrows with the common
peptides Tot1 and Tot2 as well as the specific peptides A1/2N,
A1/2S and A1/2T. The Tot1 and Tot2 are at the C-terminal of
.beta.-casein where the sequence is identical for A1 and A2 and are
not present in proteose peptones. The peptides A1/2N, A1/2S and
A1/2T include the amino acid at position 67 that differs between A1
and A2;
[0050] FIG. 15 shows the result obtained by LC-MS of a tryptic
digest of different finished products including skimmed milk powder
and several infant formulas with regard to the peptide A1S;
[0051] FIG. 16 shows the result obtained by LC-MS of a tryptic
digest of different finished products including skimmed milk powder
and several infant formulas with regard to the peptide A1N;
[0052] FIG. 17 shows the result obtained by LC-MS of a tryptic
digest of different finished products including skimmed milk powder
and several infant formulas with regard to the peptide A1T;
[0053] FIG. 18 shows the result obtained by LC-MS of a tryptic
digest of different finished products including skimmed milk powder
and several infant formulas with regard to the peptide A2S;
[0054] FIG. 19 shows the result obtained by LC-MS of a tryptic
digest of different finished products including skimmed milk powder
and several infant formulas with regard to the peptide A2N;
[0055] FIG. 20 shows the result obtained by LC-MS of a tryptic
digest of different finished products including skimmed milk powder
and several infant formulas with regard to the peptide A2T;
[0056] FIG. 21 shows the result of a tryptic digest of different
finished products including skimmed milk powder and several infant
formulas with regard to the peptide Tot1;
[0057] FIG. 22 shows the result of a tryptic digest of different
finished products including skimmed milk powder and several infant
formulas with regard to the peptide Tot2;
[0058] FIG. 23 shows the result of a tryptic digest on skimmed milk
powder, lactose, and different whey protein concentrates (WPC)
measuring the amount of the Tot1 peptide using LC-MS;
[0059] FIG. 24 shows the result of a tryptic digest on skimmed milk
powder, lactose, and different whey protein concentrates (WPC)
measuring the amount of the Tot2 peptide using LC-MS;
[0060] FIG. 25 shows the result of a tryptic digest of skimmed milk
powder, lactose and different whey protein concentrates (WPC)
measuring the amount of the A1S peptide using LC-MS;
[0061] FIG. 26 shows the result of a tryptic digest of skimmed milk
powder, lactose and different whey protein concentrates (WPC)
measuring the amount of the A1N peptide using LC-MS;
[0062] FIG. 27 shows the result of a tryptic digest of skimmed milk
powder, lactose and different whey protein concentrates (WPC)
measuring the amount of the A1T peptide using LC-MS;
[0063] FIG. 28 shows the result of a tryptic digest of skimmed milk
powder, lactose and different whey protein concentrates (WPC)
measuring the amount of the A2S peptide using LC-MS;
[0064] FIG. 29 shows the result of a tryptic digest of skimmed milk
powder, lactose and different whey protein concentrates (WPC)
measuring the amount of the A2N peptide using LC-MS;
[0065] FIG. 30 shows the result of a tryptic digest of skimmed milk
powder, lactose and different whey protein concentrates (WPC)
measuring the amount of the A2T peptide using LC-MS;
[0066] The present invention will now be described in more detail
in the following.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0067] Prior to discussing the present invention in further
details, the following terms and conventions will first be
defined:
[0068] The term "LC" refers to "liquid chromatography" as known to
the person skilled in the art. It should be emphasized that LC also
includes HPLC i.e. high performance liquid chromatography and UPLC
i.e. ultra performance liquid chromatography.
[0069] The term "MS" refers to "mass spectroscopy" as known to the
person skilled in the art. It should be emphasized that MS also
includes HRMS i.e. high resolution mass spectroscopy.
[0070] The term "compounds of defined m/z values" means that the MS
is set to measure compounds having only m/z values specifically
defined by the user when measuring on a given sample.
[0071] The term "A1/A1 cows" refers to cows with the homozygous
genotype A1A1. Milk obtained from A1/A1 cows is denoted "A1
milk".
[0072] The term "A2/A2 cows" refers to cows with the homozygous
genotype A2A2. Milk obtained from A2/A2 cows is denoted "A2
milk".
[0073] The term "A1/A2 cows" refers to cows with the heterozygous
genotype A1A2. Milk obtained from A1/A2 cows is denoted "A1/A2
milk".
[0074] The term "A1 whey" refers to whey produced essentially from
A1 milk.
[0075] The term "A2 whey" refers to whey produced essentially from
A2 milk.
[0076] The term "A1/A2 whey" refers to whey either being produced
from A1/A2 milk or being a mixture of A1 whey and A2 whey or being
produced from a mixture of A1 milk and A2 milk, A1 milk and A1/A2
milk, A2 milk and A1/A2 milk or A1 milk, A2 milk and A1/A2
milk.
[0077] The term "A2 .beta.-casein" refers to the A2 variant of
bovine .beta.-casein having the amino acid sequence according to
SEQ ID NO. 1 (secreted form of protein). In the present context,
other variants, which include a proline in position 67 may be
included into A2 .beta.-casein.
[0078] The term "A1 .beta.-casein" refers to the A1 variant of
bovine .beta.-casein having the amino acid sequence according to
SEQ ID NO. 2 (secreted form of protein). SEQ ID NO. 1 and SEQ ID
NO. 2 only differ from each other in that A1 .beta.-casein contains
a histidine at position 67, whereas A2 .beta.-casein contains a
proline at position 67. In the present context, other variants,
which include a histidine in position 67 may be included into A1
.beta.-casein.
[0079] The term "intact .beta.-casein" refers to the protein, which
is not cleaved except for the removal of the signal sequence e.g. a
protein as disclosed by SEQ ID NO. 1 and 2.
[0080] The term ".beta.-casomorphin 7" also described as "BCM-7"
refers to the peptide having the amino acid sequence
Tyr-Pro-Phe-Pro-Gly-Pro-Ile.
[0081] The term "WPC" refers to "whey protein concentrate". In this
context, WPC includes traditional WPC according to the USP
definition as well as whey reduced in lactose and whey reduced
minerals i.e. the protein level may be as low as 10% w/w.
[0082] The term "WPI" refers to "whey protein isolate" and is a
whey protein concentrate having a whey protein content on a dry
basis of not less than 90% by weight.
[0083] The term "whey protein fraction" refers to a composition
comprising whey proteins e.g. WPC and/or WPI.
[0084] The term "standard SMP" refers to "standard skimmed milk
powder" and is derived from milk obtained from mixed herds of cows
and thus, comprises multiple variants of .beta.-casein including A1
.beta.-casein and A2 .beta.-casein.
[0085] The term "A2 SMP" refers to "A2 skimmed milk powder" and
comprises only A2 .beta.-casein and not A1 .beta.-casein.
[0086] The term "proteoform" refers to highly related protein
molecules arising from all combinatorial sources of variation
giving rise to products arising from a single gene. This includes
products differing due to genetic variations, alternatively spliced
RNA transcripts and post-translational modifications.
[0087] The term "proteose peptone" is the same as the one used in
Swaisgood, 1982, i.e. the term "proteose peptone" refers to those
proteins/peptides that remain in solution after milk has been
heated at 95.degree. C. for 20 minutes and then acidified to pH 4.7
with 12% trichloro-acetic acid.
[0088] The term ".beta.-casein derived proteose peptones" refers to
proteose peptones derived from .beta.-casein alone such as PP-5,
PP-8 fast and PP-8 slow.
[0089] The term "proteose peptone 5", "PP5" or "PP-5" refers to the
residues 1-105 and 1-107 derived from .beta.-casein.
[0090] The term "proteose peptone 8 fast", "PP8f" or "PP8 fast"
refers to the residues 1-28 derived from .beta.-casein. PP8 fast
may also be referred to as "bcas4P 1-28".
[0091] The term "proteose peptone 8 slow", "PP8s" or "PP8 slow"
refers to the residues 29-105 and 29-107 derived from
.beta.-casein. PP8 slow may also be referred to as "bcas1P 29-105"
and "bcas1P 29-107".
[0092] The term "infant" refers to a child under the age of 12
months; in one embodiment of the invention the term "infant" can be
extended to include children at any age up to and including 18
months, or at any age up to and including 24 months.
[0093] The term "infant formula" as used herein refers to a
foodstuff intended for particular nutritional use by infants during
the first months of life (such as from 0 to 12 months, 0 to 10
months, 0 to 8 months, 0 to 6 months or 0 to 4 months) and
satisfying by itself the nutritional requirements of this category
of person (Article 2(c) of the European Commission Directive
91/321/EEC 2006/141/EC of 22 Dec. 2006 on infant formulae and
follow-on formulae). It also refers to a nutritional composition
intended for infants and as defined in Codex Alimentarius (Codex
STAN 72-1981) and Infant Specialities (incl. Food for Special
Medical Purpose). The expression "infant formula" encompasses both
"starter infant formula" and "follow-up formula" or "follow-on
formula".
[0094] The term "follow-up formula" or "follow-on formula" refers
to a formula that is given from the 6th month onwards. It
constitutes the principal liquid element in the progressively
diversified diet of this category of person.
[0095] The term "powder" in the present context means a dry, bulk
solid composed of a large number of very fine particles that may
flow freely when shaken or tilted. The powder may contain water in
amounts not exceeding 10%, such as not exceeding 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2%, 1.5%, 1% or 0.5%.
[0096] The term "nutritional composition" means a composition,
which nourishes a subject. This nutritional composition is usually
to be taken orally or intravenously. It may include a lipid or fat
source, a carbohydrate source and/or a protein source. The
nutritional composition of the present invention can be in solid
form (e.g. powder) or in liquid form.
[0097] In a particular embodiment, the composition of the present
invention is a hypoallergenic nutritional composition. The
expression "hypoallergenic nutritional composition" means a
nutritional composition that is unlikely to cause allergic
reactions.
[0098] In a particular embodiment, the nutritional composition of
the present invention is a "synthetic nutritional composition". The
expression "synthetic nutritional composition" means a mixture
obtained by chemical and/or biological means, which can be
chemically identical to the mixture naturally occurring in
mammalian milks (i.e. the synthetic nutritional composition is not
breast milk).
[0099] "Probiotic bacteria" means microbial cell preparations or
components of microbial cells with a beneficial effect on the
health or well-being of the host. A definition of probiotic
bacteria is given in Salminen S et al. 1999;
[0100] "Prebiotic" means a selectively fermented ingredient that
allows specific changes, both in the composition and/or activity in
the gastrointestinal microflora that confers benefits upon host
well-being and health. Prebiotica are discussed in Roberfroid M B
2007;
[0101] Detection and/or Quantification of .beta.-Casein Derived
Proteose Peptones and/or .beta.-Casein
[0102] The invention relates according to a first aspect to a
method for determining and/or quantifying .beta.-casein derived
proteose peptones and/or .beta.-casein, said method comprising the
steps of [0103] (i) providing a dairy-based product to be analysed;
[0104] (ii) subjecting said product using liquid
chromatography-mass spectrometry analysis; [0105] (iii) determining
and/or quantifying said .beta.-casein derived proteose peptones
and/or .beta.-casein in said product by detecting compounds of
defined m/z values or deconvoluting one or more mass spectrometry
spectra to calculate monoisotopic masses.
[0106] The dairy-based product may be selected from a list of
finished products such as infant formula, maternal nutrition, adult
nutritionals or dairy products such as milk, milk powder, liquid
whey, whey powder, caseinates, WPC, WPI.
[0107] In one embodiment, the dairy-based product to be analysed is
an infant formula or a whey protein fraction.
[0108] The dairy-based product to be analysed may be a powder or a
liquid. In one embodiment, the product is a powder, which is
dissolved prior to analysing the product. The powder is preferably
dissolved by dispersing the powder in water. However, the powder
may also be dissolved in other liquids such as a reducing agent as
defined below, buffered solutions such as ammonium bicarbonate,
tris, trisodium citrate, HEPES, TEAB (triethylammonium bicarbonate)
or denaturing buffers as defined below and combinations hereof.
[0109] In one embodiment, the powder is dissolved in a reducing
agent and a buffered solution, and optionally a denaturing buffer
is added subsequently. In a further embodiment, the powder is
dissolved in a reducing agent and denaturing buffer, and optionally
a buffered solution is added subsequently. In a still further
embodiment, the powder is dissolved in a buffered solution and a
denaturing buffer, and optionally a reducing agent is added
subsequently.
[0110] In one embodiment, the powders are dispersed to a
concentration of at the most 10% w/v protein in the liquid, such as
at the most 9.5% w/v protein in the liquid, like at the most 9% w/v
protein in the liquid, such as at the most 8.5 w/v % protein in the
liquid, like at the most 8% w/v protein in the liquid, such as at
the most 7.5% w/v protein in the liquid, like at the most 7% w/v
protein in the liquid, such as at the most 6.5% w/v protein in the
liquid, like at the most 6% w/v protein in the liquid, such as at
the most 5.5% w/v protein in the liquid, like at the most 5% w/v
protein in the liquid, such as at the most 4.5% w/v protein in the
liquid, like at the most 4% w/v protein in the liquid, such as at
the most 3.5 w/v % protein in the liquid, like at the most 3% w/v
protein in the liquid, such as at the most 2.5% w/v protein in the
liquid, like at the most 2% w/v protein in the liquid, such as at
the most 1.5% w/v protein in the liquid, like at the most 1% w/v
protein in the liquid, such as at the most 0.5% w/v protein in the
liquid.
[0111] After the dissolving of the samples or alternatively for
dissolving the samples, they are denatured and reduced by means of
a denaturing and reducing buffers such as urea, thiourea, guanidine
HCl, DTT, beta-mercaptoethanol, TCEP.
[0112] In one embodiment, the sample is dissolved in water and
diluted further in a mixture of trisodium citrate, Guanidine-HCl
and DTT.
[0113] In another embodiment, the sample is dissolved in Tris and
Urea and diluted in ammonium bicarbonate and DTT.
[0114] Preferably, the samples are also cleared before being
analysed. This may be performed by centrifugation. However, this
may also be performed by filtration.
[0115] In one embodiment, the product to be analysed is analysed
using intact protein analysis where the proteins are intact when
subjecting them to liquid chromatography and following mass
spectrometry (LC-MS), such as liquid chromatography high resolution
mass spectrometry (LC-HRMS). Using intact protein analysis makes is
possible to distinguish between different proteoforms of
.beta.-casein and genetic variants of full-length .beta.-casein
including the A1 and A2 variants. Thus, measurement on intact
protein from the products makes it possible to obtain a complete
fingerprinting of the protein profile of the product. Also, the
different genetic variants of proteose peptones may be
distinguished using the intact protein analysis method.
[0116] In another embodiment, said product is analysed by peptide
analysis comprising a step of enzymatic digest of said product
prior to analysing it. Thus, before being subjected to an analysis
of LC-MS the product is subjected to an enzymatic digest e.g by
digesting the product by means of trypsin or GluC (Endoproteinase
GluC [Staphylococcus aureus Protease V8]).
[0117] Using enzymatic digestion makes it possible to determine if
A1 .beta.-casein is present in a product or if proteose peptones
from A1 and/or A2 .beta.-casein are. However, due to the set-up of
the method involving digestion of the proteins as such it is not
possible to distinguish between proteose peptones and full-length
.beta.-casein but only to distinguish between the A1 and A2 genetic
variants. This may be done by detecting sequences covering the
amino acid 67, which differs between the A1 and A2 variant. Thus, a
complete fingerprinting protein profile would not be obtained but
the presence of the A1 variant and possible precursors for the
BCM-7 would be detected.
[0118] In a further embodiment, said enzymatic digest is performed
by tryptic digest or GluC digest.
[0119] In one embodiment, the liquid chromatography is a high
performance liquid chromatography. In a further embodiment, the
liquid chromatography is an ultra performance liquid
chromatography. In a still further embodiment, the liquid
chromatography is a nano liquid chromatography.
[0120] Standard setting for running the liquid chromatography
process as known to the person skilled in the art may be used in
this measurement.
[0121] In a preferred embodiment for the intact protein analysis,
the samples may be separated on a C4 column using a gradient at 0.5
ml/min. Preferably a buffer containing trifluoroacetic acid is
used. However, the gradient may vary from 0.1 ml/min to 1 ml/min,
such as from 0.1 ml/min to 0.5 ml/min. Alternatively, the following
buffers may be used for the LC separation such as water/methanol or
water/acetonitrile possibly including formic acid, difluoroacetic
acid and/or trifluoroacetic acid.
[0122] In a preferred embodiment for the peptide analysis, the
samples may be separated on a C18 column using a gradient at 75
.mu.l/min. Preferably a buffer containing formic acid is used.
However, the gradient may vary from 0.1 .mu.l/min to 100 .mu.l/min,
such as from 0.4 .mu.I/min to 85 .mu.I/min. Alternatively, the
following buffers may be used for the LC separation such as
water/methanol or water/acetonitrile possibly including formic
acid, difluoroacetic acid and/or trifluoroacetic acid.
[0123] The MS chromatograms may be recorded on machines such as
Thermo Orbitrap Elite or Thermo Q-Exactive HF.
[0124] In one embodiment, the MS chromatograms are recorded on a
Thermo Orbitrap Elite (heater temperature: 60.degree. C., sheath
gas: 20, aux gas: 5, sweep gas: 0, spray voltage: 3.8 kV, capillary
temperature: 320.degree. C., S-lens RF level: 60%, mass range 400
to 2,000 m/z, resolution 240,000, AGC target: 1e6).
[0125] In another embodiment, the MS chromatograms are recorded on
a Thermo Q-Exactive HF (heater temp: 100.degree. C., sheath gas:
53, aux gas: 14, sweep gas: 3, spray voltage: 3.5 kV, capillary
temp: 320.degree. C., S-lens RF level: 70%, mass range: 400 to
2,000 m/z, resolution: 240,000, AGC target: 1e6, maximum IT: 200
ms).
[0126] In one embodiment, said mass spectrometry analysis is a high
resolution mass spectrometry analysis.
[0127] In one embodiment, the HRMS acquisition is preferably
performed at a resolution above 100,000, such as between 120,000
and 240,000.
[0128] According to one embodiment, the determining and/or
quantifying said .beta.-casein derived proteose peptones is
performed by detecting compounds of defined m/z values as
indicated. Hereby, the presence and amount of specific peptide
sequences may be detected. This is possible as the enzymatic
digestion would lead to specific peptides due to specific
cleavage.
[0129] In one embodiment, the peptide method uses an untargeted
(data dependent acquisition-DDA). In another embodiment, the
peptide method uses an untargeted (data independent
acquisition-DIA) approach.
[0130] In a further embodiment, the peptide method uses a PRM
(parallel reaction monitoring) or a MRM (multiple reaction
monitoring) method. The precursors may preferably be selected on
the first quadrupole and fragmented and MS2 data recorded.
[0131] In one embodiment, precursor selection may be done according
to the peptide list described by SEQ ID NO. 3-10.
[0132] According to another embodiment, the mass spectrometry data
obtained after the measurement using LC-MS for each sample are
deconvoluted to calculate monoisotopic masses. Thus in one
embodiment, the determining and/or quantifying said .beta.-casein
derived proteose peptones in said product is performed by
deconvoluting one or more mass spectrometry spectra to calculate
monoisotopic masses.
[0133] The deconvolution may be performed by commercially available
software such as the Thermo BioPharma Finder.
[0134] In one embodiment, the deconvolution is performed by a
sliding windows algorithm. In a further embodiment, the
deconvolution is performed by a fixed windows algorithm.
[0135] In one preferred embodiment, the deconvolution was performed
by using Thermo BioPharma Finder 1.0 software using the Xtract
algorithm (S/N threshold: 3, relative abundance threshold: 1%, fit
factor: 80%, remainder threshold: 25%, overlaps, charge states: 5
to 50, minimum intensity: 1, expected intensity error: 3, m/z: 600
to 2000, minimum number of detected charge states: 3) and sliding
windows (time: 5 to 20 min, target average spectrum width: 0.1 min,
target average spectrum offset: 50%, merge tolerance: 1.5 Da,
maximum RT gap: 0.5 min, minimum number of detected intervals: 3,
XIC).
[0136] Deconvoluted monoisotopic masses were compared to a protein
database containing the major milk protein components .alpha.S1-CN,
.alpha.S2-CN, .beta.-CN, .kappa.-CN, .gamma.-CN,
.alpha.-lactalbumin, .beta.-lactoglobulin, CGMP and .beta.-casein
proteose peptones. Combinatorial addition of standard protein
modifications (phosphorylation, oxidation, lactosylation,
glycosylation, and pyroglutamic acid) were tested to identify the
majority of signals.
[0137] One way of quantifying the .beta.-casein derived proteose
peptones and/or the .beta.-casein is to compare the signal
intensities obtained by the measurements to a standard curve
derived from measurements of known amounts of the different
proteose peptones and/or .beta.-casein. Hereby, the absolute amount
of the proteose peptones and/or .beta.-casein in a given sample may
be calculated and the amount quantified.
[0138] Alternatively, the signal intensity of the proteose peptone
of interest may be expressed in terms of relative terms such as a
percentage of the total amount of either protein in the sample or
as a percentage of the .beta.-casein content.
[0139] The compounds to be determined and/or quantified are
.beta.-casein derived proteose peptones and/or .beta.-casein. In
one embodiment, the proteose peptones are A1 .beta.-casein derived
proteose peptones. In a further embodiment, the .beta.-casein
derived proteose peptones are PP8 fast, PP8 slow and/or PP-5. In a
still further embodiment, the A1 .beta.-casein derived proteose
peptones are PP8 fast, PP8 slow and/or PP-5.
[0140] In one embodiment, the .beta.-casein to be detected and/or
quantified is A1 .beta.-casein.
[0141] Whey Protein Fraction Having Reduced .beta.-Casein Derived
Proteose Peptone Content (Selected Whey Protein Fraction)
[0142] In a second aspect, this invention relates to a method for
producing a whey protein fraction having a reduced .beta.-casein
derived proteose peptone content, said method comprising the steps
of [0143] (i) providing a whey protein fraction; [0144] (ii)
determining and quantifying said .beta.-casein derived proteose
peptones in said whey protein fraction as described herein; and
[0145] (iii) selecting said whey protein fraction having at the
most 10% by weight of .beta.-casein derived proteose peptones based
on the total protein in said whey protein fraction forming a
selected whey protein fraction.
[0146] Available whey protein fractions to be used in finished
products such as nutritional compositions including infant formulas
often comprises A1 whey and/or A1A2 whey due to standard production
methods. However, this has not previously be considered an issue
e.g. for the manufacturing of A2 infant formula, as A1
.beta.-casein would be precipitated during the preparation of the
whey and thus, not be part of the whey protein fraction. However,
as shown in the examples, proteose peptones derived from A1
.beta.-casein is detected in whey protein fractions such as
WPC.
[0147] In one embodiment, the whey protein fraction is based upon
A1 whey and/or A1A2 whey
[0148] Due to the benefits of the method as described above for
determination and quantification of .beta.-casein variants and
proteoforms the content of .beta.-casein derived proteose peptones
may be easily detected and quantified. Hereby, the whey protein
fractions may be separated into whey protein fractions containing
different amounts of proteose peptones. Thus, whey protein
fractions may be selected, which comprises at the most 10% by
weight of proteose peptones out of the total amount of proteins in
the whey protein fraction.
[0149] In one embodiment, the selected whey protein fraction has a
.beta.-casein derived proteose peptone content, such as a
.beta.-casein derived proteose peptone content, of at the most 9.5%
by weight, such as at the most 9% by weight, preferably at the most
8.5% by weight, such as at the most 8% by weight, more preferably
at the most 7.5% by weight, such as at the most 7% by weight, even
more preferably at the most 6.5% by weight, such as at the most 6%
by weight, still more preferably at the most 5.5% by weight, such
as at the most 5% by weight, most preferably at the most 4.5% by
weight, such as at the most 4% by weight, at the most 3.5% by
weight, such as at the most 3% by weight, preferably at the most
2.5% by weight, such as at the most 2% by weight, more preferably
at the most 1.5% by weight, such as at the most 1% by weight, even
more preferably at the most 0.75% by weight, such as at the most
0.50% by weight, still more preferably at the most 0.25% by weight,
such as at the most 0.10% by weight, most preferably at the most
0.05% by weight, such as at the most 0.01% by weight, based on
total protein in the whey protein fraction.
[0150] The total amount of protein in the whey protein fraction is
preferably measured by Kjeldahl analyses (ISO 8968-1:2014).
[0151] In one embodiment, the proteose peptones are PP8 fast, PP8
slow and/or PP-5. In a further embodiment, the proteose peptones
are PP8 slow and/or PP-5.
[0152] In one embodiment, the selected whey protein fraction has a
PP8 slow and/or PP-5 content, of at the most 9.5% by weight, such
as at the most 9% by weight, preferably at the most 8.5% by weight,
such as at the most 8% by weight, more preferably at the most 7.5%
by weight, such as at the most 7% by weight, even more preferably
at the most 6.5% by weight, such as at the most 6% by weight, still
more preferably at the most 5.5% by weight, such as at the most 5%
by weight, most preferably at the most 4.5% by weight, such as at
the most 4% by weight, at the most 3.5% by weight, such as at the
most 3% by weight, preferably at the most 2.5% by weight, such as
at the most 2% by weight, more preferably at the most 1.5% by
weight, such as at the most 1% by weight, even more preferably at
the most 0.75% by weight, such as at the most 0.50% by weight,
still more preferably at the most 0.25% by weight, such as at the
most 0.10% by weight, most preferably at the most 0.05% by weight,
such as at the most 0.01% by weight, based on total protein in the
whey protein fraction.
[0153] In a still further embodiment, the proteose peptone is
PP-5.
[0154] Whey Protein Fraction Having Reduced Proteose Peptone
Content (Reduced Whey Protein Fraction)
[0155] In a third aspect, this invention relates to a method for
producing a whey protein fraction having a reduced .beta.-casein
derived proteose peptone content, said method comprising the steps
of [0156] (i) providing a whey protein fraction; [0157] (ii)
reducing the .beta.-casein derived proteose peptone content in said
whey protein fraction to a concentration of at the most 10% by
weight based on total protein in the whey protein fraction forming
a reduced whey protein fraction.
[0158] In a further aspect, this invention relates to a method for
producing a whey protein fraction having a reduced .beta.-casein
derived proteose peptone content, said method comprising the steps
of [0159] (i) providing a whey protein fraction has an initial
content of proteose peptones; [0160] (ii) reducing the
.beta.-casein derived proteose peptone content in said whey protein
fraction to a concentration of at the least 5.times. less than the
initial content of said whey protein fraction obtaining a reduced
whey protein fraction, such as at least 8.times. less, like at the
least 10.times. less, such as at the least 15.times. less than the
initial content of said whey protein fraction.
[0161] In one embodiment, said whey protein fraction is based upon
A1 whey and/or A1A2 whey.
[0162] By means of example, the at least 5.times. less is to be
understood as if for example the initial content is 5 .mu.g/mg then
a 5.times. (five times) reduction would mean that the reduced whey
protein fraction contains at the most 1 .mu.g/mg and similarly if
the signal intensity is 500 000 000 for the initial content then
the signal intensity would be at the most 100 000 000 for the
reduced whey protein fraction.
[0163] The reduction of at least 5.times. less, 8.times. less,
10.times. less or 15.times. less may be measured by means of the
signal intensity obtained by the method for determining and
quantifying as described herein.
[0164] In one embodiment, the whey protein fraction is tested using
the method as described herein for determining and quantifying the
content of proteose peptones in order to decide whether or not the
content of proteose peptones needs to be reduced or not before the
whey protein fractions is to be used.
[0165] The content of the .beta.-casein derived proteose peptones,
in the whey protein fraction is reduced to at the most 10% by
weight based on the total protein in the whey protein fraction.
[0166] The total amount of protein in the whey protein fraction is
preferably measured by Kjeldahl analyses (ISO 8968-1:2014).
[0167] In one embodiment, the proteose peptone content is reduced
by gel filtration. However, the proteose peptone content may also
be reduced by means of ion exchange chromatography, affinity
chromatography or separation over membranes.
[0168] In a further embodiment, the final content of proteose
peptones in the reduced whey protein fraction may be determined and
quantified by the method as described above.
[0169] In one embodiment, the reduced .beta.-casein derived whey
protein fraction has a proteose peptone content of at the most 9.5%
by weight, such as at the most 9% by weight, preferably at the most
8.5% by weight, such as at the most 8% by weight, more preferably
at the most 7.5% by weight, such as at the most 7% by weight, even
more preferably at the most 6.5% by weight, such as at the most 6%
by weight, still more preferably at the most 5.5% by weight, such
as at the most 5% by weight, most preferably at the most 4.5% by
weight, such as at the most 4% by weight, at the most 3.5% by
weight, such as at the most 3% by weight, preferably at the most
2.5% by weight, such as at the most 2% by weight, more preferably
at the most 1.5% by weight, such as at the most 1% by weight, even
more preferably at the most 0.75% by weight, such as at the most
0.50% by weight, still more preferably at the most 0.25% by weight,
such as at the most 0.10% by weight, most preferably at the most
0.05% by weight, such as at the most 0.01% by weight, based on
total protein in the whey protein fraction.
[0170] In one embodiment, the proteose peptones are PP8 fast, PP8
slow and/or PP-5. In a further embodiment, the proteose peptones
are PP8 slow and/or PP-5.
[0171] In one embodiment, the reduced whey protein fraction has a
PP8 slow and/or PP-5 content of at the most 9.5% by weight, such as
at the most 9% by weight, preferably at the most 8.5% by weight,
such as at the most 8% by weight, more preferably at the most 7.5%
by weight, such as at the most 7% by weight, even more preferably
at the most 6.5% by weight, such as at the most 6% by weight, still
more preferably at the most 5.5% by weight, such as at the most 5%
by weight, most preferably at the most 4.5% by weight, such as at
the most 4% by weight, at the most 3.5% by weight, such as at the
most 3% by weight, preferably at the most 2.5% by weight, such as
at the most 2% by weight, more preferably at the most 1.5% by
weight, such as at the most 1% by weight, even more preferably at
the most 0.75% by weight, such as at the most 0.50% by weight,
still more preferably at the most 0.25% by weight, such as at the
most 0.10% by weight, most preferably at the most 0.05% by weight,
such as at the most 0.01% by weight, based on total protein in the
whey protein fraction.
[0172] In a still further embodiment, the proteose peptone is
PP-5.
[0173] Nutritional Composition Such as an Infant Formula Having
Reduced Proteose Peptone Content
[0174] In a fourth aspect, the invention relates to a method for
producing a nutritional composition having a reduced .beta.-casein
derived proteose peptone content, said method comprising the steps
of [0175] (i) providing a selected whey protein fraction or a
reduced whey protein fraction as described herein; [0176] (ii)
preparing said nutritional composition with a reduced proteose
peptone content from said selected whey protein fractions, said
reduced whey protein fractions or a mixture hereof.
[0177] In a fifth aspect, the invention relates to a whey protein
fraction having a reduced .beta.-casein derived proteose peptone
content, obtainable by the method as described herein.
[0178] In a sixth aspect, the invention relates to a nutritional
composition comprising said whey protein fraction having a reduced
.beta.-casein derived proteose peptone content as described herein
or is obtainable by the method as described herein.
[0179] In a further embodiment, the .beta.-casein derived proteose
peptone content of the nutritional composition such as an infant
formula is at the most 9% by weight based on total protein in the
nutritional composition.
[0180] The total amount of protein in the whey protein fraction is
preferably measured by Kjeldahl analyses (ISO 8968-1:2014).
[0181] In a still further embodiment, the .beta.-casein derived
proteose peptone content of the nutritional composition is at the
most 8.5% by weight, more preferably at the most 8% by weight, such
as at the most 7.5% by weight, even more preferably at the most 7%
by weight, such as at the most 6.5% by weight, still more
preferably at the most 6% by weight, such as at the most 5.5% by
weight, most preferably at the most 5% by weight, such as at the
most 4.5% by weight, like at the most 4% by weight, at the most
3.5% by weight, more preferably at the most 3% by weight, such as
at the most 2.5% by weight, even more preferably at the most 2% by
weight, such as at the most 1.5% by weight, still more preferably
at the most 1% by weight, such as at the most 0.75% by weight, most
preferably at the most 0.50% by weight, such as at the most 0.25%
by weight, like at the most 0.1% by weight, such as at the most
0.05% by weight, like at the most 0.01% by weight, based on total
protein in the whey protein fraction.
[0182] The nutritional composition of the invention may be intended
for any mammal, such as for example humans, and pets such as cats
and dogs. In a preferred embodiment, the mammal is a human.
[0183] Examples of nutritional compositions are infant formula,
maternal nutrition, adult nutritionals or dairy products.
[0184] In one embodiment, the nutritional composition is an infant
formula.
[0185] The general composition of a nutritional composition such as
an infant formula for use according to the present invention may
optionally contain substances which may have a beneficial effect
such as probiotic bacteria, fibres, lactoferrin, nucleotides,
nucleosides, and/or the like in the amounts such as those
customarily found in nutritional compositions to be fed to
infants.
[0186] The probiotic bacteria may be selected from the group
consisting of Lactobacillus such as Lactobacillus rhamnosus,
Lactobacillus paracasei and Lactobacillus reuteri and
Bifidobacterium such as Bifidobacterium lactis, Bifidobacterium
breve and Bifidobacterium longum.
[0187] The nutritional composition, such as the infant formula, may
optionally further comprise prebiotics, such as nondigestable
carbohydrates that promote the growth of probiotic bacteria in the
gut.
[0188] In a preferred embodiment, nutritional composition, such as
the infant formula, comprises prebiotics selected from the group
consisting of fructooligosaccharides (FOS), raftilose, inulin,
raftiline, lactulose, cows' milk oligosaccharides (CMOS) and
galactooligosaccharides (GOS).
[0189] The nutritional composition may also contain all vitamins
and minerals understood to be essential in the daily diet in
nutritionally significant amounts.
[0190] Thus a preferred embodiment relates to a nutritional
composition, such as an infant formula, further comprising
vitamins.
[0191] Minimum requirements have been established for certain
vitamins and minerals. Examples of minerals, vitamins and other
nutrients optionally present in the nutritional composition include
vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin
E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin,
biotin, pantothenic acid, choline, calcium, phosphorous, iodine,
iron, magnesium, copper, zinc, manganese, chloride, potassium,
sodium, selenium, chromium, molybdenum, taurine, and L-carnitine.
Minerals are usually added in salt form.
[0192] If necessary, the nutritional composition, such as the
infant formula, may contain emulsifiers and stabilizers such as soy
lecithin, citric acid esters of mono- and di-glycerides, and the
like. This is especially the case if the composition is provided in
liquid form.
[0193] A preferred embodiment relates to a nutritional composition,
such as an infant formula, wherein at least one source of
carbohydrates is selected form the group consisting of lactose,
corn syrup solids, fructose, glucose, maltodextrins, dried glucose
syrups, sucrose, trehalose, galactose, maltose, honey powders,
starch, oligosaccharides, raftiline and raftilose.
[0194] Another preferred embodiment relates to a nutritional
composition, such as an infant formula, further comprising
anhydrous milk fat.
[0195] A yet other preferred embodiment relates to a nutritional
composition, such as an infant formula, further comprising LC-PUFAs
such as DHA, EPA, DPA and/or ARA.
[0196] The nutritional composition, such as the infant formula, may
further comprise flavours such as but not limited to vanillin.
[0197] A particular preferred embodiment relates to a nutritional
composition, such as an infant formula, further comprising
fructo-oligosaccharides such as raftiline and/or raftilose.
[0198] The nutritional composition, such as the infant formula, may
be a liquid formula or a powder that is to be reconstituted before
use. A preferred embodiment is an infant formula being a
powder.
[0199] In a preferred embodiment the nutritional composition is a
liquid composition that has been prepared by reconstituting a
powder.
[0200] The present invention further provides a nutritional
composition, such as an infant formula, according to the present
invention for use as complementary feeding of infants in
association with human breast milk.
[0201] According to particular embodiments the infant is at an age
between 0-12 months, preferably at an age between 0-6 months.
[0202] Therapeutic and Non-Therapeutic Uses of a Nutritional
Composition or Infant Formula Having a Reduced Proteose Peptone
Content.
[0203] In a seventh aspect of the present invention, the
nutritional composition as described herein can be used as a
medicament.
[0204] In an eight aspect of the present invention, the infant
formula as described herein relates to the use in treating,
preventing and/or ameliorating abdominal pain in an infant.
[0205] In a ninth aspect of the present invention, the infant
formula as described herein relates to the use in treating,
preventing and/or ameliorating lactose intolerance in an
infant.
[0206] A tenth aspect of the present invention relates to the use
of the infant formula as described herein for improving the stool
consistency.
[0207] BCM-7 is known to affect the opioid receptors and is related
to the stool consistency and abdominal pain. Thus, an infant
formula comprising none or only a very limited amount of BCM-7
precursors i.e. proteose peptones PP-5 and PP8 slow derived from A1
.beta.-casein, would result in less BCM-7 formed during the
consumption of the infant formula and thus, less disadvantageous
influence on the stool consistency and abdominal pain.
[0208] Furthermore, it is believed that administration of an infant
formula of containing less precursor molecules of the BCM-7 and
consequently resulting in less BCM-7 formed will result in less
influence on opioid receptors being affected preventing lactose
intolerance later in that infant's life. There is growing evidence
that events early in life can precipitate disease, even though the
disease may only manifest later in life.
[0209] In this context, treating is to be understood as a situation
where a disorder is established, in contrast to preventing which
takes place prior to a disorder is established. Ameliorating is to
be understood as a situation where a disorder is established but
not treated in a manner which results in the disorder disappearing
but mere improves the health conditions of the individual suffering
from the disease.
[0210] In another aspect, the present invention relates to a method
of treating, preventing and/or ameliorating abdominal pain in an
infant.
[0211] In a further aspect, the present invention relates to a
method of treating, preventing and/or ameliorating lactose
intolerance in an infant.
[0212] It should be noted that embodiments and features described
in the context of one of the aspects of the present invention also
apply to the other aspects of the invention.
[0213] All patent and non-patent references cited in the present
application, are hereby incorporated by reference in their
entirety.
[0214] The invention will now be described in further details in
the following non-limiting examples.
EXAMPLES
[0215] Material and Methods
[0216] Chemical Reagents and Samples
[0217] Guanidine hydrochloride (RDD001), trisodium citrate
dihydrate (S1804), DL-dithiothreitol (43819) and .beta.-casein
standard (C6905) were purchased from Sigma-Aldrich (St. Louis,
Mich., USA), TFA (1.08178.0050) from VWR International (Radnor,
Pa., USA), and LC-MS grade water (1.15333.1000) and acetonitrile
(1.00029.1000) from Merck (Darmstadt, Germany).
[0218] Milk Samples
[0219] Raw milk was collected from the Teagasc Moorepark dairy farm
(Fermoy, Co. Cork, Ireland) from cows that were genetically
profiled as A1/A1, defatted, pasteurized using a Microthermics
(UHT/HTST Electric Model 25HV Hybrid, Liquid Technologies, Wexford,
Ireland) unit heated to 85.degree. C. and held for 23 s, followed
by homogenization [GEA Niro Soavi S.p.A. Type: NS2006H
(non-aseptic)] using 2-stage homogenization at a total
homogenization pressure of 2500 psi. The sample was spray dried to
produce a skimmed milk powder sample referred to as A1|A1 SMP.
Commercially available A1/A2 and A2/A2 SMP were purchased from
Europe and the USA, buffalo SMP was purchased from India, and
pasteurized breast milk from pooled human donors (991-01-P) was
purchased from Lee Biosolutions (Maryland Heights, Mo. USA).
Different batches, either from the same or different manufacturers,
are numbered A, B, C etc.
[0220] Finished Products and Whey Protein Concentrates
[0221] Commercially available infant formula powders suitable for
infants (0-12 months) and children (1 year and above) were obtained
from different manufacturers and for different age groups. The
different samples were denoted either IF1-IF22 (see Table 1) or
IFa-IFj in the experiments. In this regard, it should be noted that
IFa=IF1, IFb=IF2, IFc=IF3, IFd=IF4, IFe=IF5, IFf=IF6 and
IFg=IF7.
TABLE-US-00001 TABLE 1 ID Type Stage % protein % casein* %
.beta.-casein** IF1 A2 1 10.2 35 1.19 IF2 A2 2 15.3 60 3.06 IF3 A2
3 13.9 60 2.78 IF4 A2 4 17.0 60 3.40 IF5 A2 1 10.4 30 1.04 IF6 A2 2
15.0 50 2.50 IF7 A2 3 15.0 60 3.00 IF8 A1/A2 1 10.6 40 1.41 IF9
A1/A2 2 14.9 40.sctn. 1.99 IF10 A1/A2 3 15.3 70.sctn. 3.57 IF11
A1/A2 3 22.2 78 5.77 IF12 A1/A2 1 11.0 40 1.47 IF13 A1/A2 1 10.2 35
1.25 IF14 A1/A2 2 15.3 60 3.02 IF15 A1/A2 3 15.0 60 3.07 IF16 A1/A2
4 17.0 60 3.48 IF17 A2 1 9.9 30 1.01 IF18 A2 2 10.4 30 1.03 IF19 A2
3 12.8 50 1.94 IF20 A2 1 10.1 35 1.32 IF21 A2 2 11.7 60 2.41 IF22
A2 3 15.2 60 3.12 *as indicated on the label (except in .sctn.,
which are based on assumptions), **calculated by multiplying the
protein content by the casein content and dividing by 3 (as stated
in Swaisgood, 1995)
[0222] Three different commercially available batches (denoted A, B
and C) of WPC were tested. WPC35 is a whey protein concentrate with
35% w/w protein.
[0223] Two different commercially available batches of WPC28
(denoted A and B) were tested. WPC28 is demineralized WPC with 28%
w/w protein.
[0224] WPC80 is a commercially available whey protein concentrate
with 80% w/w protein which is alpha-lactalbumin enriched.
[0225] Sample Preparation
[0226] Powders were dispersed to 3.5% (w/v) protein in water using
a volumetric flask, stirred for at least 30 min at room
temperature, denatured with 4 volumes of denaturing buffer
(Guanidine HCl 7.5 M, trisodium citrate 6.25 mM, DTT 23 mM),
incubated at room temperature for 30 min, and cleared by
centrifugation at 16,000 g for 10 min.
[0227] Trypsin Digestion of Intact Proteins
[0228] The sample powders were dispersed to 3.5% (w/v) protein in
Tris 50 mM+6M urea by mixing on an orbital shaker for one hour.
[0229] 200 .mu.L sample where diluted with 1000 .mu.L ammonium
bicarbonate 100 mM and vortex'ed. 10 .mu.L of DTT (45 mM) was added
to 100 .mu.L of the diluted solution and incubated at 60.degree. C.
for 30 minutes. The sample was then cooled to room temperature
before quickly spinning the sample and adding 10 .mu.L
iodoacetamide (100 mM). The sample was then incubated for 30
minutes at room temperature in the dark before adding 6 .mu.L
trypsin (0.2 .mu.g/.mu.L) and incubating overnight at 37.degree. C.
The tryptic digestion is stopped by adding 6 .mu.L formic acid 10%.
The digested sample is centrifuged at 14,000 g for 10 minutes
before transferring the liquid phase into a vial for injection and
determination via LC-MS.
[0230] LC-MS Analysis
[0231] Cleared samples were analyzed by LC-MS based on existing
methods (Bonfatti et al., 2008, Frederiksen et al., 2011).
[0232] For the intact protein analysis, samples were separated on a
C4 column (Acquity UPLC Protein BEH C4, 300 .ANG., 1.7 .mu.m, 2.1
mm.times.150 mm) using the following gradient at 0.5 ml/min (Table
2):
TABLE-US-00002 TABLE 2 Time (min) 0 4 7 22 23 24 25 30 % Buffer B
15 35 42.5 52.5 80 80 15 15 Buffer A: 0.1% trifluoroacetic acid
(TFA) in water, Buffer B: 0.1% TFA in acetonitrile: water 90:10
[0233] The MS signal was recorded from 4 to 20 min in Full MS mode
on a Thermo Q-Exactive HF (heater temp: 100.degree. C., sheath gas:
53, aux gas: 14, sweep gas: 3, spray voltage: 3.5 kV, capillary
temp: 320.degree. C., S-lens RF level: 70%, mass range: 400 to
2,000 m/z, resolution: 240,000, AGC target: 1e6, maximum IT: 200
ms).
[0234] For the peptide analysis i.e. tryptic digest or GluC digest,
the peptides were separated on a C18 column (Acquity UPLC BEH C18,
130 .ANG., 1.7 .mu.m, 1.0.times.150 mm) using the following
gradient at 75 .mu.L/min (Table 3):
TABLE-US-00003 TABLE 3 Time (min) 0 30 31 33 35 45 % Buffer B 2 60
100 100 2 2
[0235] The MS signal was recorded from 3.5 to 35 min in PRM mode on
a Thermo Q-Exactive HF (heater temp: 30.degree. C., sheath gas: 8,
aux gas: 0, sweep gas: 0, spray voltage: 3.6 kV, capillary temp:
320.degree. C., S-lens RF level: 55, default charge: 2, MS2
resolution: 30,000, AGC target: 1e5, maximum IT: 100 ms, isolation
window: 1.5 m/Z, isolation offset: 0.5 m/z).
[0236] The inclusion list was as shown in Table 4:
TABLE-US-00004 TABLE 4 SEQ ID Name Mass [m/z] Formula +M+ NO A1N
884.95449 AQTQSLVYPFPGPIHN 3 A2N 864.95141 AQTQSLVYPFPGPIPN 4 Tot2
415.7296 AVPYPQR 5 A1S 755.06013 IHPFAQTQSLVYPFPG 6 PIHN A1T
1072.17772 IHPFAQTQSLVYPFPG 7 PIHNSLPQNIPPLTQT PWVPPFLQPEVMGVSK A2S
1112.08349 IHPFAQTQSLVYPFPG 8 PIPN A2T 1329.9688 IHPFAQTQSLVYPFPG 9
PIPNSLPQNIPPLTQT PWVPPFLQPEVMGVSK Tot1 390.75254 VLPVPQK 10
[0237] Data Deconvolution
[0238] Raw data files were deconvoluted using Thermo BioPharma
Finder 1.0 software using the Xtract algorithm (S/N threshold: 3,
relative abundance threshold: 1%, fit factor: 80%, remainder
threshold: 25%, overlaps, charge states: 5 to 50, minimum
intensity: 1, expected intensity error: 3, m/z: 600 to 2000,
minimum number of detected charge states: 3) and sliding windows
(time: 5 to 20 min, target average spectrum width: 0.1 min, target
average spectrum offset: 50%, merge tolerance: 1.5 Da, maximum RT
gap: 0.5 min, minimum number of detected intervals: 3, XIC).
[0239] Monoisotopic masses, total signal intensities and apex RTs
were exported as csv files and used for the next steps.
[0240] Limit of detection experiments were also performed by
deconvoluting raw data files using a fixed window centered around
the 8-casein peak. Parameters were as follows: S/N threshold: 1,
relative abundance threshold: 1%, fit factor: 25%, remainder
threshold: 25%, overlaps, charge states: 12 to 30, minimum
intensity: 1, expected intensity error: 3, m/z: 800 to 2000,
minimum number of detected charge states: 3, time: 11 to 13.5 min,
relative intensity threshold: 1%. Monoisotopic masses and total
signal intensities were used for the next steps.
[0241] Proteoform Attribution
[0242] Deconvoluted monoisotopic masses were compared to a protein
database containing the major milk protein components .alpha.S1-CN,
.alpha.S2-CN, .beta.3-CN, .kappa.-CN, .gamma.-CN,
.alpha.-lactalbumin, .beta.-lactoglobulin, CGMP and proteose
peptones using a Nestle developed software (Protein Analyzer).
Combinatorial addition of standard protein modifications
(phosphorylation, oxidation, lactosylation, glycosylation, and
pyroglutamic acid) were tested to identify the majority of signals.
Misattributed signals were corrected by manual verification. Total
signal intensities were extracted for the relevant proteins or
proteoforms (i.e. total .beta.-casein, .beta.-casein lactosylation
states, .beta.-casein genetic variants etc.). For clarity, only
.beta.-casein genetic variants A1, A2 and B are detailed in the
Figures. .beta.-casein I/H2 co-elutes with .beta.-casein and was
merged with .beta.-casein A2. Both variants have a proline at
position 67 and belong to the A2 type.
[0243] Solid-State Protein Glycation
[0244] The solid-state glycation experiment was based on Fenaille,
2003 (Fenaille et al., 2003). Briefly, 45 g of milk powder were
incubated in an enclosure saturated with dipotassium carbonate for
8-10 days to reach 5.4% humidity (initial % was 3.8%). Nine 2.1
g-aliquots of the humidified powder were incubated in 25-mL glass
tubes at 60.degree. C. in an oven for 45 min to 24 h. Glycation was
stopped by transferring the tubes on ice for 5 min. Then, 20 g of
water were added to obtain a 3.5% (w/v) protein solution, which was
mixed at room temperature for 1 h 30 on a roller mixer. Solutions
were stored at 4.degree. C. overnight until the end of the
experiment and warmed up for 2 h at RT before analysis. Samples
were prepared as described above except that the incubation step
was 10 min at 60.degree. C. on a thermomixer (650 rpm). MS
chromatograms were recorded on a Thermo Orbitrap Elite (heater
temperature: 60.degree. C., sheath gas: 20, aux gas: 5, sweep gas:
0, spray voltage: 3.8 kV, capillary temperature: 320.degree. C.,
S-lens RF level: 60%, mass range: 400 to 2,000 m/z, resolution:
240,000, AGC target: 1e6).
[0245] .beta.-Casein Quantification
[0246] A skim milk powder (SMP) sample was injected multiple times
throughout each analytical series to normalize the signals across
experiments.
[0247] An external calibration curve was established using
.beta.-casein standard adjusted for protein purity (see text).
.beta.-casein signal intensities were normalized with the average
.beta.-casein signal of the SMP samples (corrected for glycation,
see above). The calibration curve was forced to 0 (.gamma.=0.0524x,
where x is expressed in .mu.g .beta.-casein injected and .gamma. is
dimensionless).
[0248] For each sample, .beta.-casein intensity values were first
corrected for glycation and normalized with the average
.beta.-casein signal of the SMP samples (corrected for glycation).
Then the amount of .beta.-casein injected was calculated through
the external calibration curve. This calculation can be done for
total .beta.-casein, for a given variant e.g. A1 .beta.-casein, or
for a set of variants (.beta.-casein A2 results also include the
related I/H2 variant).
[0249] Method performance was evaluated by two operators in seven
infant formula samples (stages 1-4, manufactured using skim milk
powder of the A2 type) using triplicate injections on five
different days.
Example 1--LC-MS Method, Data Output, Deconvolution and
Visualization
[0250] To analyze intact proteins, infant formulas and skim milk
powder samples were dispersed in water to 3.5% (v/w) protein,
denatured in 6 M guanidine, reduced with DTT, and cleared by
centrifugation. Intact proteins were separated by UPLC on a C4
column using a water/ACN gradient with 0.1% TFA and analyzed by
high resolution mass spectrometry (FIG. 3A). Chromatograms are
sequentially deconvoluted using sliding windows and the Xtract
algorithm to obtain the monoisotopic masses of the proteins (FIG.
3B). Finally, proteoforms are visualized on bubble charts, with
their retention time on the X-axis, monoisotopic size on the
Y-axis, and signal intensity as bubble area (FIG. 3C).
Example 2--Global Fingerprinting
[0251] FIG. 4A illustrates method output with cow raw milk and
highlights where major milk proteins (.alpha.S1-, .alpha.S2-,
.beta.-, .kappa.- and .gamma.-casein, .alpha.-lac and .beta.-Ig)
are detected. Zooming into the .beta.-casein region (FIG. 4B)
allows detailed analysis of the various .beta.-casein proteoforms
including genetic variants and protein modification
(process-induced lactosylated adducts are detectable in all skim
milk powder samples). Mass accuracy is shown in Table 5 for the raw
milk sample. A 1 Da difference may be due to a misattributed
monoisotopic signal propagated throughout the deconvolution since a
merging tolerance of 1.5 Da is applied between the sliding windows
to avoid splitting signals into two masses that are 1 Da apart. The
method can be used to profile a wide variety of raw materials and
finished products (FIG. 5). Interestingly, this method readily
detects casein glycomacropeptide (CGMP), the C-terminus part of
.kappa.-casein released by rennet enzyme during cheesemaking
process, a polypeptide notoriously hard to detect by gel staining
or UV profiling.
TABLE-US-00005 TABLE 5 mass accuracy in cow raw milk sample
expected detected .DELTA.mass in ppm .beta.-casein A1 (5P) 24008.16
24007.00 -1.16 -48.51 .beta.-casein A2 (5P) 23968.15 23967.10 -1.06
-44.09 .alpha.S1-casein(8P) 23600.21 23599.64 -0.57 -24.18
.alpha.S2-casein(11P) 25212.98 25212.12 -0.86 -34.17 .alpha.-lac
14176.81 14176.87 0.06 4.44 .beta.-lg A 18355.46 18355.54 0.08 4.09
.beta.-lg B 18269.42 18269.49 0.07 3.78 .kappa.-casein A 19025.53
19025.62 0.09 4.85 .kappa.-casein B 18993.58 18993.73 0.15 7.86
.gamma.2-casein 11816.25 11816.25 0.00 -0.42 .gamma.3-casein
11551.10 11551.13 0.03 2.60
Example 3--Detection of .beta.-Casein A1 in .beta.-Casein A2 Infant
Formulas
[0252] Seven .beta.-casein A2 infant formulas were spiked with
A1|A1 SMP to 5% .beta.-casein A1 (5 g .beta.-casein A1 in 100 g of
total .beta.-casein). At this concentration, .beta.-casein A1
signal was detected in all cases (FIG. 6).
Example 4--Protein Quantification and Glycation
[0253] Protein quantification requires not only a suitable standard
to establish a calibration curve but also that different
proteoforms exhibit similar signal responses. This is necessary to
apply the calibration curve to unknown samples given that the
standard and the samples are likely to have different proteoform
distributions. Alternatively, the method should provide a way to
correct for the proteoform distribution.
[0254] To explore whether protein lactosylation influences MS
signal intensity, glycation was induced in a skim milk powder
sample by heating as outlined in the materials and methods (FIG.
7A). The increase in glycated .beta.-casein (FIG. 7B) was
accompanied by a decrease in the total .beta.-casein signal (FIG.
7C). Plotting the total .beta.-casein signal against the percentage
of unglycated .beta.-casein signal followed a linear relationship
(FIG. 7D) that was converted into a fold signal reduction (FIG. 7E)
of the form .gamma.=1/(ax+b), with a and b corresponding to 0.878
and 0.122, respectively. For .beta.-casein, a Glycation Correction
Factor (GCF) can thus be calculated from the percentage of
unglycated .beta.-casein (% UG.sub..beta.) using Equation 1.
G .times. C .times. F = 1 0 . 8 .times. 78 .times. % .times.
.times. UG .beta. + 0 . 1 .times. 2 .times. 2 ( Equation .times.
.times. 1 ) ##EQU00001##
[0255] Multiplying the measured .beta.-casein signal by the GCF
yields the corrected .beta.-casein signal (.beta.cas.sub.corr),
which corresponds to the expected signal if all .beta.-casein were
unglycated (Equation 2).
.beta.cas.sub.corr=.beta.cas.sub.meas.times.GCF (Equation 2)
Example 5--.beta.-Casein Quantification
[0256] Quantification was assessed using the sliding windows
algorithm. An external calibration curve was established using
commercial purified .beta.-casein. This standard also contained
.alpha.S1-casein, .kappa.-casein, and various unidentified peptides
and protein fragments, most of which were matching .beta.-casein
cleavage products (FIG. 8A). The fraction of the total signal
attributable to intact .beta.-casein in the seven most concentrated
calibration samples reached 85.6.+-.0.4%, and the concentration of
standard .beta.-casein was adjusted with this purity factor. For
each concentration, the total .beta.-casein signal was normalized
to that of the average (and corrected for glycation) signal of SMP
samples that had been injected in the same series (and in all
subsequent quantification experiments to account for variations in
MS performance), and normalized values were plotted against the
amount of .beta.-casein loaded on the column. The calibration curve
(FIG. 8B) showed good linearity (R.sup.2=0.996) with a random
distribution of the residuals. The amount of .beta.-casein injected
on the column (in .mu.g) can be calculated from this calibration
curve using Equation 3:
.beta. .times. c .times. a .times. s i .times. n .times. j
.function. [ g ] = .beta. .times. c .times. a .times. s c .times. o
.times. r .times. r , n .times. o .times. r .times. m 0 . 0 .times.
5 .times. 2 .times. 4 ( Equation .times. .times. 3 )
##EQU00002##
[0257] where .beta.cas.sub.corr,norm is the corrected signal for
.beta.-casein in the sample (Equation 2) divided by the average of
the corrected signals for the skim milk powder samples injected in
the same series (Equation 4).
.beta. .times. c .times. a .times. s c .times. o .times. r .times.
r , n .times. o .times. r .times. m = .beta. .times. c .times. a
.times. s c .times. o .times. r .times. r .beta. _ .times. cas S
.times. M .times. P , c .times. o .times. r .times. r ( Equation
.times. .times. 4 ) ##EQU00003##
[0258] Quantification of .beta.-casein variants in a dairy matrix
was further assessed by mixing a skim milk powder containing only
the A1 variant with a skim milk powder containing only the A2 type
(FIG. 8C). The response for .beta.-casein A2 showed good linearity
(R.sup.2=0.996), indicating that the matrix effect was negligible.
The measured and expected A1/A2 ratios showed a very strong linear
relationship (% A2.sub.calc=1.05% A2.sub.exp, R.sup.2=0.996). This
means that the response factors of both variants are very close and
suggests that the calibration curve is also suitable to quantify
individual genetic variants. Quantification of .beta.-casein in the
two SMP samples yielded an average value of 28.3 g
.beta.-casein/100 g protein, in line with the 27% value described
in the Handbook of milk composition (Swaisgood, 1995) (Table
6).
TABLE-US-00006 TABLE 6 Quantification of .beta.-casein in A1|A1 and
A2|A2 SMP MS signal normalized to SMP .mu.g .beta.-casein on column
A2|A2 A1|A1 SMP (n = 3) A2|A2 A1|A1 A2|A2 A1|A1 Rep1 389956856
311884671 336558582 1.16 0.93 22.1 17.7 Rep2 374319489 319123478
336558582 1.11 0.95 21.2 18.1 Average 382138173 315504074 336558582
1.14 0.94 21.7 17.9 .mu.g protein on column 70.0 70.0 .beta.-casein
content 31% 26%
Example 6--Method Performance (.beta.-Casein Quantification)
[0259] Method performance was assessed in seven infant formulas
containing only .beta.-casein A2. Each sample was prepared five
different times and analyzed by triplicate injection. The measured
amount of 8-casein was generally in line (83%-136%) with
theoretical values calculated by multiplying the protein content by
the casein content and by 33%, which represents the generally
accepted proportion of .beta.-casein in the total casein
(Swaisgood, 1995) (FIG. 9A).
[0260] The method was further tested on another series of
commercially available infant formulas (either A2-based or
manufactured with standard SMP) and shown to be generally in line
with the theoretical values (FIG. 9B, the % whey is based on
assumptions for hatched values). The differences between measured
and theoretical .beta.-casein content were the largest for samples
with the smallest 8-casein content. This might be due to the fact
that the less abundant proteoforms become too close to the noise to
be measured effectively, in particular in samples with multiple
genetic variants such as IF8 and IF13.
Example 7--Detection of A1 Specific Proteose Peptones in Whey
Protein Concentrate by Intact Protein Analysis
[0261] The six different batches of whey protein concentrates where
analysed using the intact protein analysis as described in the
methods and materials section using LC-HRMS.
[0262] Raw material analysed: [0263] Three batches of WPC35 [0264]
Two batches of WPC28 [0265] One batch of WPC80 (.alpha.-lac
enriched)
[0266] FIG. 10 illustrates the separate analyses of the six
different whey protein concentrates.
[0267] None of the WPC tested showed traces of 8-casein as no
signals were detected within the circle with the solid perimeter
(i.e circle in the upper right corner).
[0268] In contrast, all WPCs tested showed signals corresponding to
proteose peptones detected as signals within the circle with the
dashed perimeter (i.e. circles in the lower left corner).
[0269] In addition, the analysis showed that all ingredients
contained CGMP (casein glycomacropeptide), which is a
casein-derived peptide, identified in the lower left corner of each
of the diagrams. Just below the circle with the dashed
perimeter
[0270] FIG. 11 is a close-up of the area within the circle with the
dashed perimeter shown in FIG. 10. This close-up demonstrates that
signals corresponding to the expected masses for a large variety of
proteose peptones were found in all ingredients. Black represents
.beta.-casein A2 derived proteose peptones, gray represents
.beta.-casein A1 derived proteose peptones and white represent
unassigned compounds;
[0271] Some of these peptones are: PP5 A1 1-105, PP5 A1 1-107, PP5
A2 1-105, PP5 A2 1-107, bcas1P A1 29-105 (i.e. PP8s A1 29-105),
bcas1P A1 29-107 (i.e. PP8s A1 29-107), bcas1p A2 29-105 (i.e. PP8s
A2 29-105) and bcas1P A2 29-107 (PP8s A2 29-107).
[0272] No gamma-casein was detected in the WPC tested.
[0273] WPC80 did not contain PP8 slow (29-105/7). Probably due to
the ultrafiltration step in the process of preparing WPC80, which
would likely remove PP8 slow.
[0274] Both in FIG. 10 and FIG. 11 several signals (shown in white)
both within and outside of the circle with the dashed perimeter in
FIG. 10 relates to peptides remaining identification.
[0275] Accordingly, proteose peptones from .beta.-casein A1 (and
A2) were found in all whey raw material tested. Hence, they will be
present in finished products and are extremely likely to be
responsible for the high A1 signal picked up in all finished
products tested.
Example 8--Detection of A1 Specific Proteose Peptones in Finished
Products by Intact Protein Analysis
[0276] Different finished products being different infant formulas
of different brands were analysed for the detection of specific
proteose peptones. All of the infant formulas are considered A2
finished products.
[0277] The finished products were dissolved and analysed by LC-HRMS
as described in the materials and methods section above.
[0278] FIG. 12 shows that proteose peptones were found in all of
the tested batches even though the proteose peptone profile
differed among the finished products.
[0279] A1 .beta.-casein derived proteose peptones are discovered in
all of the infant formulas tested even though they are based on
milk with A2 .beta.-casein.
Example 9--Quantification of A1 and A2 Specific Proteose Peptones
by Intact Protein Analysis
[0280] Relative quantification of PP5 A1 and PP5 A2 in a dairy
matrix was assessed by mixing a skim milk powder containing only
the A1 variant with a skim milk powder containing only the A2 type
(FIG. 13). The responses for PP5 A2 and PP5 A1 showed good dose
response having quadratic regression (R.sup.2=0.999 and
R.sup.2=0.995, respectively), indicating that the matrix effect was
negligible. This means that the response factors of both variants
are very close and suggests that the calibration curve is also
suitable to quantify individual genetic variants.
[0281] Conclusively, it is shown that specific proteose peptones
can be quantified using the intact protein analysis even in a
matrix.
Example 10--Detection of A1 specific proteose peptones in finished
products by tryptic digest
[0282] Different finished products being either standard skimmed
milk powder (i.e. standard SMP), skimmed milk powder containing
only the A2 version of .beta.-casein or different infant formulas
of different brands and for different age-groups. All of the infant
formulas are considered A2 finished products.
[0283] The finished products were dispersed to 3.5% (w/v) protein
in Tris 50 mM+6M urea and mixed on an orbital shaker for one hour.
200 .mu.L sample were diluted with 1000 .mu.L ammonium bicarbonate
100 mM and vortexed. To 100 .mu.L of the diluted solution, 10 .mu.L
of DTT 45 mM were added. Samples were incubated at 60.degree. C.
for 30 minutes, cooled to RT and quickly spun to collect
evaporation droplets. 10 .mu.L iodoacetamide 100 mM (in ammonium
bicarbonate 100 mM) were added; samples were incubated 30 minutes
at room temperature in the dark. 6 .mu.L trypsin 0.2 .mu.g/.mu.L
were added and samples were incubated overnight at 37.degree. C. 6
.mu.L formic acid 10% were added to stop digestion, solutions were
centrifuged at 14,000 g for 10 minutes; the liquid phase was
transferred into a vial for injection and analysed by LC-MS as
described in the materials and methods section above.
[0284] The analysis focused on the detection of A1S peptide i.e. an
A1-specific peptide having a position as illustrated in FIG. 14. As
evident from the position of the A1S peptide it can be
distinguished from the A2S peptide as it covers the area of
.beta.-casein including the amino acid 67.
[0285] FIG. 15 illustrates that no A1S is detected in A2 SMP
samples. A1S was detected in one A2 SMP sample, which proved
subsequently to mistakenly contain .beta.-casein A1. However, the
peptide is significantly expressed in standard SMP. Surprisingly, a
strong signal was detected for the peptide A1S in all the infant
formulas tested. Similar results were obtained for the peptides A1N
(FIG. 16) and A1T (FIG. 17).
[0286] As expected the peptides A2S (FIG. 18), A2N (FIG. 19), A2T
(FIG. 20), Tot 1 (FIG. 21) and Tot 2 (FIG. 22) showed strong
signals in all of the areas tested.
Example 11--Detection of A1 Specific Proteose Peptones in Whey
Protein Concentrate by Tryptic Digest
[0287] Different batches of whey protein concentrates where
analysed together with skimmed milk powder both standard and
skimmed milk powder containing only the A2 version of
.beta.-casein.
[0288] Raw materials (RM) analysed: [0289] Three batches of WPC35
[0290] Two batches of WPC28 [0291] One batch of WPC80 (.alpha.-lac
enriched) [0292] Two different commercially available lactose
ingredients
[0293] The products were dissolved and exposed to tryptic digestion
before being analysed by LC-MS as described in the materials and
methods section above.
[0294] The analyses focused on the detection of Tot1 and Tot2
peptides i.e. common to .beta.-casein to A1 and A2, A1S peptide
i.e. an A1-specific peptide, A1N peptide i.e. an A1-specific
peptide, A1T peptide i.e. an A1-specific peptide, A2N peptide i.e.
an A2-specific peptide, A2T peptide i.e. an A2-specific peptide and
A2S peptide i.e. an A2-specific peptide. The position of the
peptides is illustrated in FIG. 14.
[0295] FIGS. 23 and 24 illustrate the results obtained when
determining the amount of Tot1 and Tot2 peptide, respectively. It
is a common (to A1 and A2) peptide but also a peptide present only
in intact .beta.-casein and .gamma.-casein. It is shown that the
signal for the common .beta.-casein peptide is severely reduced in
all whey ingredients, and absent in lactose ingredients but present
in the SMP and A2 SMP as would be expected.
[0296] This indicates that very little intact .beta.-casein or
.gamma.-casein is present in the RM (.about.1%) compared to SMP
even when an equivalent protein content was used, as expected.
[0297] FIG. 25 illustrates the results obtained when determining
the amount of A1S peptide. It is shown that the signal for the A1S
peptide is strongest for the SMP but lacking for A2 SMP as
expected. Also the lactose samples does not show any signal. For
the whey protein samples, a surprising amount of about a fourth of
the amount observed in SMP was determined.
[0298] Similar results were obtained when the samples were tested
for the presence of two other A1-specific peptides A1N and A1T
(FIG. 26 & FIG. 27).
[0299] FIG. 28 illustrates the results obtained when determining
the amount of A2S. It is shown that the signal for the A2S
(A2-specific) peptide is strongest for the A2 SMP but weaker for
the SMP as expected. The lactose samples do not show any signal as
expected. For the whey protein samples, a surprising amount of
about a fourth of the amount observed in SMP was determined.
[0300] Similar results were obtained when the samples were tested
for the presence of two other A2-specific peptides A2N and A2T
(FIGS. 29 and 30).
[0301] Accordingly, it can be concluded from the above results that
in all whey RM analyzed, the signal intensity of the .beta.-casein
common peptide is severely reduced (.about.100.times.) as compared
to SMP. Also, the A1- and A2-specific peptides are only
.about.4-5.times. reduced compared to SMP. Hence, there is a
selective enrichment of the A1- and A2-specific peptides.
[0302] Thus, using the tryptic digest method and determining the
different peptide, it would seem that the whey ingredients still
contain .about.20% of .beta.-casein fragments (compared to SMP),
which may at least partly be known proteose peptones such as PP5
and PP8s.
Example 12--Reduction of Proteose Peptones in Whey Protein
Fractions
[0303] In order to obtain a reduced whey protein fraction, three
batches of WPC35 where the presence of proteose peptones were
determined in Example 9, are subjected to a gel filtration.
[0304] The three batches of WPC35 are further purified by gel
filtration on a column (550.times.22 mm) of Sephadex G-75 made up
in and equilibrated with volatile 0.1 M NH.sub.4HCO.sub.3 (pH
8.0-8.5) buffer.
[0305] The 0.5 g-0.7 g of each of the batches are dissolved in 5-7
ml buffer (with addition of a few ml of 1 M NaOH to neutralize
residual traces of TCA and aid dissolution) and applied to the
column. Flow rate is adjusted to 0.5 ml/min and 5 ml fractions are
collected.
[0306] The fractions are analysed by the detection and
quantification method as described herein i.e. LC-HRMS according to
the materials and methods section. Fractions without proteose
peptones PP5 and PP8 slow are pooled to form a reduced whey protein
fraction.
REFERENCES
[0307] Bonfatti, V. et al. J Chromatogr A, 2008; 1195(1-2):101-106.
[0308] de Jong et al. J Chromatogr A, 1993; 652(1):207-213. [0309]
EFSA Scientific Report, Scientific Report of EFSA prepared by a
DATEX Working Group on the potential health impact of
b-casomorphins and related peptides. 2009; 231; 1-107. [0310]
Fenaille, F. et al. Rapid Commun Mass Spectrom, 2003;
17(13):1483-1492. [0311] Fenaille, F. et al. International Dairy
Journal, 2006; 16(7):728-739. [0312] Feng, P. et al. J AOAC Int,
2017; 100(2):510-521. [0313] Frederiksen, P. D. et al. J Dairy Sci,
2011; 94(10):4787-4799. [0314] Ho, S. et al. Eur. J. Clin. Nutr.
2014, 68, 994-1000. [0315] Karamoko, G. et al. Biotechnologie,
Agronomie, Societe et Environnement, 2013; 17(2):373-382. [0316]
Poulsen, N. A et al. Acta Agriculturae Scandinavica, Section
A--Animal Science, 2016; 66(4):190-198. [0317] Roberfroid M B. J
Nutr. 2007; 137: 830S. [0318] Salminen S et al. Trend Food Sci.
Technol., 1999; 10 107-110. [0319] Swaisgood, H. E. Handbook of
Milk Composition. R. G. Jensen, ed. Academic Press, San Diego 1995;
464-468. [0320] Swaisgood. Developments in Dairy Chemistry. Fox
(Ed), Proteins, vol. 1, 1982; 63-110. [0321] Vallejo-Cordoba, B. J
Capillary Electrophor, 1997; 4(5):219-224. [0322] Visser, S. et al.
J Chromatogr A, 1995; 711(1):141-150.
[0323] Items
[0324] 1. A method for determining and/or quantifying .beta.-casein
derived proteose peptones and/or .beta.-casein, said method
comprising the steps of [0325] (i) providing a dairy-based product
to be analysed; [0326] (ii) subjecting said product using liquid
chromatography-mass spectrometry analysis; [0327] (iii) determining
and/or quantifying said .beta.-casein derived proteose peptones
and/or .beta.-casein in said product by detecting compounds of
defined m/z values or deconvoluting one or more mass spectrometry
spectra to calculate monoisotopic masses.
[0328] 2. The method according to item 1, wherein said proteose
peptones are A1 .beta.-casein derived proteose peptones.
[0329] 3. The method according to item 2, wherein said
.beta.-casein derived proteose peptones are PP8 fast, PP8 slow
and/or PP-5, such as A1 .beta.-casein derived proteose peptones are
PP8 fast, PP8 slow and/or PP-5.
[0330] 4. The method according to any of the preceding items,
wherein said product is an infant formula or a whey protein
fraction.
[0331] 5. The method according to any of the preceding items,
wherein the product is a powder, said powder being dissolved prior
to analysing said product.
[0332] 6. The method according to any of the preceding items,
wherein said product is analysed by intact protein analysis.
[0333] 7. The method according to item 6, wherein said mass
spectrometry analysis is a high-resolution mass spectrometry
analysis.
[0334] 8. The method according to any of the items 6-7, wherein
determining and/or quantifying said .beta.-casein derived proteose
peptones in said product is performed by deconvoluting one or more
mass spectrometry spectra to calculate monoisotopic masses.
[0335] 9. The method according to item 8, wherein said
deconvolution is performed by a sliding windows algorithm.
[0336] 10. The method according to any of the items 1-5, wherein
said product is analysed by peptide analysis comprising a step of
enzymatic digest of said product prior to analysing it.
[0337] 11. The method according to item 10, wherein said enzymatic
digest is performed by tryptic digest or GluC digest.
[0338] 12. The method according to any of the items 10-11, wherein
determining and/or quantifying said .beta.-casein derived proteose
peptones is performed by detecting compounds of defined m/z
values.
[0339] 13. A method for producing a whey protein fraction having a
reduced .beta.-casein derived proteose peptone content, said method
comprising the steps of [0340] (i) providing a whey protein
fraction; [0341] (ii) determining and quantifying said
.beta.-casein derived proteose peptones in said whey protein
fraction as described in any of the items 1-12; and [0342] (iii)
selecting said whey protein fraction having at the most 10% by
weight of .beta.-casein derived proteose peptones based on the
total protein in said whey protein fraction forming a selected whey
protein fraction.
[0343] 14. A method for producing a whey protein fraction having a
reduced .beta.-casein derived proteose peptone content, said method
comprising the steps of [0344] (i) providing a whey protein
fraction; [0345] (ii) reducing the .beta.-casein derived proteose
peptone content in said whey protein fraction to a concentration of
at the most 10% by weight based on total protein in the whey
protein fraction forming a reduced whey protein fraction.
[0346] 15. The method according to item 14 further comprising the
step before and/or after step (ii) of determining and quantifying
said .beta.-casein derived proteose peptones in said whey protein
fraction as described in any one of the items 1-12.
[0347] 16. The method according to any of the items 14-15, wherein
the proteose peptone content is reduced by gel filtration.
[0348] 17. The method according to any of the items 13-16, wherein
said selected or reduced whey protein fraction has a .beta.-casein
derived proteose peptone content of at the most 9.5% by weight,
such as at the most 9% by weight, preferably at the most 8.5% by
weight, such as at the most 8% by weight, more preferably at the
most 7.5% by weight, such as at the most 7% by weight, even more
preferably at the most 6.5% by weight, such as at the most 6% by
weight, still more preferably at the most 5.5% by weight, such as
at the most 5% by weight, most preferably at the most 4.5% by
weight, such as at the most 4% by weight, at the most 3.5% by
weight, such as at the most 3% by weight, preferably at the most
2.5% by weight, such as at the most 2% by weight, more preferably
at the most 1.5% by weight, such as at the most 1% by weight, even
more preferably at the most 0.75% by weight, such as at the most
0.50% by weight, still more preferably at the most 0.25% by weight,
such as at the most 0.10% by weight, most preferably at the most
0.05% by weight, such as at the most 0.01% by weight, based on
total protein in the whey protein fraction.
[0349] 18. The method according to any of the items 13-17, wherein
said proteose peptones are PP8 fast, PP8 slow and/or PP-5.
[0350] 19. A method for producing a nutritional composition having
a reduced .beta.-casein derived proteose peptone content, said
method comprising the steps of [0351] (i) providing a selected whey
protein fraction or a reduced whey protein fraction as described in
any of the items 13-18; [0352] (ii) preparing said nutritional
composition with a reduced proteose peptone content from said
selected whey protein fractions, said reduced whey protein
fractions or a mixture hereof.
[0353] 20. A whey protein fraction having a reduced .beta.-casein
derived proteose peptone content, obtainable by the method
according to any of items 14-18.
[0354] 21. A nutritional composition comprising said whey protein
fraction having a reduced .beta.-casein derived proteose peptone
content according to item 20 or obtainable by the method according
to item 19.
[0355] 22. The nutritional composition according to item 21,
wherein said nutritional composition is an infant formula.
[0356] 23. The nutritional composition according to any of the
items 21-22, wherein said proteose peptone content is at the most
9% by weight based on total protein in the nutritional
composition.
[0357] 24. A nutritional composition according to any of items
21-23 for use as a medicament.
[0358] 25. An infant formula according to any of items 22-23 for
use in treating, preventing and/or ameliorating abdominal pain in
an infant.
[0359] 26. An infant formula according to any of items 22-23 for
use in treating, preventing and/or ameliorating lactose intolerance
in an infant.
[0360] 27. Use of the infant formula according to any of items
22-23 for improving the stool consistency.
TABLE-US-00007 Sequence listing SEQ ID NO. 1 (amino acid sequence
of A2 .beta.-casein): RELEELNVPG EIVESLSSSE ESITRINKKI EKFQSEEQQQ
TEDELQDKIH PFAQTQSLVY PFPGPIPNSL PQNIPPLTQT PVVVPPFLQP EVMGVSKVKE
AMAPKHKEMP FPKYPVEPFT ESQSLTLTDV ENLHLPLPLL QSWMHQPHQP LPPTVMFPPQ
SVLSLSQSKV LPVPQKAVPY PQRDMPIQAF LLYQEPVLGP VRGPFPIIV SEQ ID NO.2
(amino acid sequence of A1 .beta.-casein): RELEELNVPG EIVESLSSSE
ESITRINKKI EKFQSEEQQQ TEDELQDKIH PFAQTQSLVY PFPGPIHNSL PQNIPPLTQT
PVVVPPFLQP EVMGVSKVKE AMAPKHKEMP FPKYPVEPFT ESQSLTLTDV ENLHLPLPLL
QSWMHQPHQP LPPTVMFPPQ SVLSLSQSKV LPVPQKAVPY PQRDMPIQAF LLYQEPVLGP
VRGPFPIIV SEQ ID NO.3 (amino acid sequence of A1N): AQTQSLVYPF
PGPIHN SEQ ID NO.4 (amino acid sequence of A2N): AQTQSLVYPF PGPIPN
SEQ ID NO.5 (amino acid sequence of Tot2): AVPYPQR SEQ ID NO.6
(amino acid sequence of A1S): IHPFAQTQSL VYPFPGPIHN SEQ ID NO.7
(amino acid sequence of A1T): IHPFAQTQSL VYPFPGPIHN SLPQNIPPLT
QTPVVVPPFL QPEVMGVSK SEQ ID NO.8 (amino acid sequence of A2S):
IHPFAQTQSL VYPFPGPIPN SEQ ID NO.9 (amino acid sequence of A2T):
IHPFAQTQSL VYPFPGPIPN SLPQNIPPLT QTPVVVPPFL QPEVMGVSK SEQ ID NO.10
(amino acid sequence of Tot1): VLPVPQK
Sequence CWU 1
1
101209PRTUnknownamino acid sequence of A2 beta-casein 1Arg Glu Leu
Glu Glu Leu Asn Val Pro Gly Glu Ile Val Glu Ser Leu1 5 10 15Ser Ser
Ser Glu Glu Ser Ile Thr Arg Ile Asn Lys Lys Ile Glu Lys 20 25 30Phe
Gln Ser Glu Glu Gln Gln Gln Thr Glu Asp Glu Leu Gln Asp Lys 35 40
45Ile His Pro Phe Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly
50 55 60Pro Ile Pro Asn Ser Leu Pro Gln Asn Ile Pro Pro Leu Thr Gln
Thr65 70 75 80Pro Val Val Val Pro Pro Phe Leu Gln Pro Glu Val Met
Gly Val Ser 85 90 95Lys Val Lys Glu Ala Met Ala Pro Lys His Lys Glu
Met Pro Phe Pro 100 105 110Lys Tyr Pro Val Glu Pro Phe Thr Glu Ser
Gln Ser Leu Thr Leu Thr 115 120 125Asp Val Glu Asn Leu His Leu Pro
Leu Pro Leu Leu Gln Ser Trp Met 130 135 140His Gln Pro His Gln Pro
Leu Pro Pro Thr Val Met Phe Pro Pro Gln145 150 155 160Ser Val Leu
Ser Leu Ser Gln Ser Lys Val Leu Pro Val Pro Gln Lys 165 170 175Ala
Val Pro Tyr Pro Gln Arg Asp Met Pro Ile Gln Ala Phe Leu Leu 180 185
190Tyr Gln Glu Pro Val Leu Gly Pro Val Arg Gly Pro Phe Pro Ile Ile
195 200 205Val2209PRTUnknownamino acid sequence of A1 beta-casein
2Arg Glu Leu Glu Glu Leu Asn Val Pro Gly Glu Ile Val Glu Ser Leu1 5
10 15Ser Ser Ser Glu Glu Ser Ile Thr Arg Ile Asn Lys Lys Ile Glu
Lys 20 25 30Phe Gln Ser Glu Glu Gln Gln Gln Thr Glu Asp Glu Leu Gln
Asp Lys 35 40 45Ile His Pro Phe Ala Gln Thr Gln Ser Leu Val Tyr Pro
Phe Pro Gly 50 55 60Pro Ile His Asn Ser Leu Pro Gln Asn Ile Pro Pro
Leu Thr Gln Thr65 70 75 80Pro Val Val Val Pro Pro Phe Leu Gln Pro
Glu Val Met Gly Val Ser 85 90 95Lys Val Lys Glu Ala Met Ala Pro Lys
His Lys Glu Met Pro Phe Pro 100 105 110Lys Tyr Pro Val Glu Pro Phe
Thr Glu Ser Gln Ser Leu Thr Leu Thr 115 120 125Asp Val Glu Asn Leu
His Leu Pro Leu Pro Leu Leu Gln Ser Trp Met 130 135 140His Gln Pro
His Gln Pro Leu Pro Pro Thr Val Met Phe Pro Pro Gln145 150 155
160Ser Val Leu Ser Leu Ser Gln Ser Lys Val Leu Pro Val Pro Gln Lys
165 170 175Ala Val Pro Tyr Pro Gln Arg Asp Met Pro Ile Gln Ala Phe
Leu Leu 180 185 190Tyr Gln Glu Pro Val Leu Gly Pro Val Arg Gly Pro
Phe Pro Ile Ile 195 200 205Val316PRTUnknownamino acid sequence of
A1N 3Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly Pro Ile His
Asn1 5 10 15416PRTUnknownamino acid sequence of A2N 4Ala Gln Thr
Gln Ser Leu Val Tyr Pro Phe Pro Gly Pro Ile Pro Asn1 5 10
1557PRTUnknownamino acid sequence of Tot2 5Ala Val Pro Tyr Pro Gln
Arg1 5620PRTUnknownamino acid sequence of A1S 6Ile His Pro Phe Ala
Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly1 5 10 15Pro Ile His Asn
20749PRTUnknownamino acid sequence of A1T 7Ile His Pro Phe Ala Gln
Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly1 5 10 15Pro Ile His Asn Ser
Leu Pro Gln Asn Ile Pro Pro Leu Thr Gln Thr 20 25 30Pro Val Val Val
Pro Pro Phe Leu Gln Pro Glu Val Met Gly Val Ser 35 40
45Lys820PRTUnknownamino acid sequence of A2S 8Ile His Pro Phe Ala
Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly1 5 10 15Pro Ile Pro Asn
20949PRTUnknownamino acid sequence of A2T 9Ile His Pro Phe Ala Gln
Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly1 5 10 15Pro Ile Pro Asn Ser
Leu Pro Gln Asn Ile Pro Pro Leu Thr Gln Thr 20 25 30Pro Val Val Val
Pro Pro Phe Leu Gln Pro Glu Val Met Gly Val Ser 35 40
45Lys107PRTUnknownamino acid sequence of Tot1 10Val Leu Pro Val Pro
Gln Lys1 5
* * * * *